Solubility tags for the expression and purification of bioactive peptides

Abstract

Peptide tags, referred to here as inclusion body tags, are disclosed useful for the generation of insoluble fusion peptides. The fusion peptides comprise at least one inclusion body tag operably linked to a peptide of interest. Expression of the fusion peptide in a host cell results in a product that is insoluble and contained within inclusion bodies in the cell and/or cell lysate. The inclusion bodies may then be purified and the protein of interest may be isolated after cleavage from the inclusion body tag.

1. A nucleic acid molecule encoding a fusion peptide comprising an inclusion body tag operably linked to at least one peptide of interest; wherein the inclusion body tag comprises the structure:
 SEQ ID NO: 58-Spacer-[[SEQ ID NO: 58]-[Spacer] m ] n    
 wherein 
 
       
         
           
                 
               
                   SEQ ID NO: 58 is 
                 
                 
               
                   Gln-Gln-Xaa1-Phe-Xaa2-Trp-Xaa3-Phe-Xaa4-Xaa5-Gln; 
                 
             
                
               
            
             
                
               
            
           
         
         Xaa1=Arg, His, or Lys; 
         Xaa2=Gln, His, or Lys; 
         Xaa3=Gln, His, or Lys; 
         Xaa4=Glu or Gln; 
         Xaa5=Gln or Lys; 
         n=1 to 10; 
         m=n−1; and 
         Spacer=is a peptide comprising the amino acids selected from the group consisting of proline, arginine, glycine, glutamic acid, and cysteine. 
       
     
     
       2. An expression cassette comprising the nucleic acid molecule of  claim 1 . 
     
     
       3. A vector comprising the expression cassette of  claim 2 . 
     
     
       4. A microbial host cell comprising the vector of  claim 3 . 
     
     
       5. The microbial host cell of  claim 4 , wherein the host cell is selected from the group consisting of  Aspergillus, Trichoderma, Saccharomyces, Pichia, Yarrowia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella , and  Myxococcus.    
     
     
       6. A method for expressing a peptide in insoluble form comprising:
 a) synthesizing an expressible genetic construct encoding a fusion peptide comprising a first portion encoding an inclusion body tag operably linked to a second portion encoding a peptide of interest; 
 b) transforming an expression host cell with the genetic construct of (a); 
 c) growing the transformed host cell of (b) under conditions wherein the expressible genetic construct is expressed and the encoded fusion peptide is produced in an insoluble form; and 
 d) recovering said fusion peptide in said insoluble form; wherein the inclusion body tag comprises the structure: 
 SEQ ID NO: 58-Spacer-[[SEQ ID NO: 58]-[Spacer] m ] n    
 wherein 
 
       
         
           
                 
               
                   SEQ ID NO: 58 is 
                 
                 
               
                   Gln-Gln-Xaa1-Phe-Xaa2-Trp-Xaa3-Phe-Xaa4-Xaa5-Gln; 
                 
             
                
               
            
             
                
               
            
           
         
         Xaa1=Arg, His, or Lys; 
         Xaa2=Gln, His, or Lys; 
         Xaa3=Gln, His, or Lys; 
         Xaa4=Glu or Gln; 
         Xaa5=Gln or Lys; 
         n=1 to 10; 
         m=n−1; and 
         Spacer=is a peptide comprising the amino acids selected from the group consisting of proline, arginine, glycine, glutamic acid, and cysteine. 
       
     
     
       7. A method for the production of a peptide of interest comprising:
 a) synthesizing a genetic construct encoding a fusion peptide comprising a first portion encoding an inclusion body tag operably linked to a second portion encoding at least one peptide of interest; wherein said first portion and said second portion are separated by at least one cleavable peptide linker; 
 b) transforming an expression host cell with the genetic construct of (a); 
 c) growing the transformed host cell of (b) under conditions wherein the genetic construct is expressed and the encoded fusion peptide is produced in an insoluble form; 
 d) recovering the fusion peptide in said insoluble form; 
 e) cleaving said fusion peptide said at least one cleavable peptide linker whereby said first portion of the fusion peptide is no longer fused to said second portion; and 
 f) recovering said peptide of interest; wherein the inclusion body tag comprises the structure: 
 SEQ ID NO: 58-Spacer-[[SEQ ID NO: 58]-[Spacer] m ] n    
 wherein 
 
       
         
           
                 
               
                   SEQ ID NO: 58 is 
                 
                 
               
                   Gln-Gln-Xaa1-Phe-Xaa2-Trp-Xaa3-Phe-Xaa4-Xaa5-Gln; 
                 
             
                
               
            
             
                
               
            
           
         
         Xaa1=Arg, His, or Lys; 
         Xaa2=Gln, His, or Lys; 
         Xaa3=Gln, His, or Lys; 
         Xaa4=Glu or Gln; 
         Xaa5=Gln or Lys; 
         n=1 to 10; 
         m=n−1; and 
         Spacer=is a peptide comprising the amino acids selected from the group consisting of proline, arginine, glycine, glutamic acid, and cysteine. 
       
     
     
       8. The method according to  claim 7  wherein the cleavable peptide linker is cleaved using chemical hydrolysis, enzymatic hydrolysis, or combinations thereof. 
     
     
       9. The method according to  claim 7  wherein the peptide of interest is selected from the group consisting of a polymer-binding peptide, a hair-binding peptide, a nail-binding peptide, a skin-binding peptide, a clay-binding peptide, a pigment-binding peptide, a cellulose-binding peptide, and an antimicrobial peptide. 
     
     
       10. The method according to  claim 9  wherein the hair-binding peptide is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 7, 9, 11, 13, 23, and 59-147. 
     
     
       11. The method according to  claim 9  wherein the skin-binding peptide is selected from the group consisting of SEQ ID NOs: 148 to 155. 
     
     
       12. The method according to  claim 9  wherein the nail-binding peptide is selected from the group consisting of SEQ ID NOs: 156 to 157. 
     
     
       13. The method according to  claim 9  wherein the polymer binding peptide is selected from the group consisting of SEQ ID NOs: 218 to 244. 
     
     
       14. The method according to  claim 9  wherein the clay-binding peptide is selected from the group consisting of SEQ ID NOs: 245 to 260. 
     
     
       15. The method according to  claim 9  wherein the antimicrobial peptide is selected from the group consisting of SEQ ID NOs: 158 to 186.